CN111381042A

CN111381042A - Prediction method of large blood vessel invasion in hepatocellular carcinoma, kit and application thereof

Info

Publication number: CN111381042A
Application number: CN201811641289.XA
Authority: CN
Inventors: 方雷; 杜荣辉; 王忠夏
Original assignee: Nanjing University
Current assignee: Nanjing University
Priority date: 2018-12-29
Filing date: 2018-12-29
Publication date: 2020-07-07
Anticipated expiration: 2038-12-29
Also published as: CN111381042B

Abstract

The invention finds protein markers specific to HCC patients with different degrees of vascular invasion, and further provides a method for predicting the large vessel invasion of hepatocellular carcinoma (HCC) patients. The invention also develops a detection kit for the large vessel invasion of hepatocellular carcinoma (HCC) patients, which can be used for immunoblot and immunohistochemical analysis, and the diagnosis and grading of the blood vessel invasion condition can be carried out at the early stage of HCC occurrence through the detection of the kit, and an optimized treatment scheme is proposed for the treatment of the HCC, so that the incidence of HCC large vessel invasion is reduced, and the prognosis and survival rate of HCC are improved.

Description

Prediction method of large blood vessel invasion in hepatocellular carcinoma, kit and application thereof

Technical Field

The invention relates to a prediction method of large blood vessel invasion in hepatocellular carcinoma.

Background

Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related death worldwide, resulting in about 600,000 deaths per year. Therapeutic hepatectomy is now widely recognized as the first treatment of HCC with good liver function reserve, particularly early stage HCC. Unfortunately, approximately 70% of HCC patients relapse within 5 years after radical hepatectomy. Vascular invasion is generally considered to be an important risk factor for the prognosis of HCC patients after hepatectomy. Vascular invasion of hepatocellular carcinoma can be further classified as microvascular and macrovascular invasion. Microvascular invasion (MVI) is defined as tumor migration into the vascular space surrounded by endothelium, identifiable only under microscopy, while macrovascular invasion (MAV) refers to the invasion of a large amount of tumor into the main trunk or branches of portal or hepatic veins. MVI and MAV may represent a stepwise process of vascular invasion, ultimately leading to intrahepatic spread and distant metastasis of HCC. Multiple studies have shown that vascular invasion, including MVI and MAV, is negatively correlated with the survival of HCC patients. MVI patients can benefit from anatomic hepatectomy, transcatheter treatment, and radiation therapy. Despite recent improvements in diagnostic and therapeutic regimens, the prognosis of large vessel invasion of HCC remains daunting. Median survival in patients with MAV HCC is reported to be only 2-6 months with supportive care. MAV has traditionally been considered a contraindication to surgical resection, although there are several inconsistent studies reporting that the survival rates of HCC and MAV are improved by radical surgical procedures. International guidelines, including the barcelona clinical liver cancer system, european liver disease research association and asia pacific liver research association, suggest sorafenib as the only choice for advanced HCC and MAV. However, sorafenib only slightly improved survival by 1-3 months. At the time of diagnosis, about 10-40% of HCC patients present MAV in the liver and/or portal vein. The reported incidence of MAV is low when HCC is diagnosed early, while MAV is found in up to 44% of patients with end-stage HCC. Studies have shown that MAV is positively correlated with malignancy and mortality in HCC. Therefore, it is urgent and necessary to find more promising molecular markers for diagnosing the vascular invasion condition of HCC at an early stage. Meanwhile, the current molecular mechanism research on the occurrence of MAV is quite limited, and the elucidation of the potential mechanism of MAV occurrence in HCC is of great significance, which may lead to the innovation of the treatment method, thereby improving the prognosis of this fatal disease. Nitrogen (Nitrogen) is an essential component of all proteins, RNA and DNA of the human body. The urea cycle is an essential pathway for nitrogen metabolism in the liver, and consists of 5 metabolic enzymes CPS1, OTC, ASS1, ARG1 and ASL, and is used for detoxifying ammonia and converting highly toxic ammonia into urea to be excreted to the outside of the body. There have been several studies that have shown that in many cancerous tumors, key enzymes in the urea cycle are down-regulated or inactivated, thereby increasing the reuse of waste ammonia, which is converted to amino acids again, and even used to synthesize pyrimidines to support the synthesis of cancer cell RNA and DNA, ultimately aiding the growth and proliferation of the tumor. However, although the urea cycle has been reported to be associated with the development of a variety of cancers, including HCC, its exact function in HCC with large vessel invasion remains unclear.

In recent years, precision medicine based on multigroup science techniques has been developed. Genomics, sequencing and transcriptomics, proteomics, and metabolomics technologies are widely used in clinical diagnosis and elucidation of disease pathogenesis. Among them, proteomics is a major target of various therapeutic modalities as well as a major performer of life activities due to its study target protein, and thus has attracted great attention. The discovery and identification of disease-associated marker proteins, signaling pathways and metabolic pathways from large-cohort clinical patient samples through proteomic approaches combined with in-depth bioinformatic analysis has become a hot spot and important approach in current basic medical and clinical research.

Disclosure of Invention

Hepatocellular carcinoma (HCC) is one of the most fatal cancers in the world. Vascular invasion reflects the aggressiveness of HCC and is considered a key risk factor for tumor recurrence. In the present invention, to better understand the molecular mechanisms of macrovascular invasion and metastasis in HCC patients and to find protein markers specific for vascular invasion, we performed high throughput proteomics studies based on iTRAQ technology to determine proteins that are significantly dysregulated in the HCC patient's cancer tissues of MVI (-), MVI (+) and MAV. In HCC patients with MAV, we found that 47 proteins were significantly down-regulated in their tumor tissues; whereas 30 of these proteins were unchanged in the tumor tissues of HCC patients with MVI (-) and MVI (+). These proteins, especially key enzymes in the urea cycle, as specific clinically relevant proteomic features in patients with large vessel invasion HCC, can be used for precise diagnosis of MAV and provide potential therapeutic targets for treatment of MAV.

To achieve the above objects, the present invention provides a method for predicting macrovascular invasion in a hepatocellular carcinoma (HCC) patient, when one or more specific protein markers, which are proteins found to be dysregulated in the HCC patient with MAV based on high-throughput proteomic studies, are dysregulated, i.e., expression levels are up-or down-regulated.

In one embodiment, the upregulation refers to a fold change greater than 1.2 fold and the downregulation refers to a fold change less than 0.8 fold.

In one embodiment, the specific protein marker dysregulation is the down-regulated expression of one or more of the proteins numbered P25325, P05062, P00167, P52758, Q93099, P04424, P11586, Q13228, P08319, P30084, P34913, Q6UX53, Q9UI17, P169930, P00966, P05089, P22033, A6NLP5, P55084, P42765, P07127, O75891, P05091, P09467, Q16822, Q53FZ2, Q9UBR1, Q93088, P00505, P54868, P36871, P35573, P80404, Q3LXA3, P06737, Q9Y2P5, Q50251, P33121, P16825, P08133, P23141, O08154, P231957, P95867, Q0628678, Q06247, Q9Y2P 08147, Q3851, P33133121, P16825, P08178, P2317, P8647, and Q8647.

In one embodiment, deregulated expression level of said specific protein marker is an occurrence of down-regulation of the expression level of one or more of CPS1, ASS1, ASL, ARG1, BHMT, DMGDH, Annexin a6 and CES 1.

In one embodiment, the specific protein marker deregulation is the upregulation of the expression level of CKAP 4.

In one embodiment, the specific protein marker deregulation is the down-regulation of the expression level of one or more of the 5 key enzymes of the urea cycle, CPS1, OTC, ASS1, ARG1 and ASL.

In one embodiment, the specific protein marker deregulation is the down-regulation of the expression level of one or more of the 5 metabolic enzymes of the urea cycle, BHMT2, DMGDH, SARDH and GLDC.

In one embodiment, a hepatocellular carcinoma (HCC) patient is predicted to develop macrovascular invasion when the patient presents a urea cycle disorder associated with down-regulation of the expression levels of 5 key enzymes of the urea cycle, CPS1, OTC, ASS1, ARG1 or ASL, or of the expression levels of 5 metabolic enzymes of the urea cycle, BHMT2, DMGDH, SARDH or GLDC.

In one embodiment, the specific protein marker is determined by:

(1) obtaining cancer tissues and corresponding paracancerous tissues of a hepatocellular carcinoma patient suffering from vascular invasion by means of surgery or biopsy puncture;

(2) extracting total protein from different patient tissues by Bioruptor non-contact ultrasonic crushing with high efficiency;

(3) reducing and alkylating the obtained protein, loading the protein into an ultrafiltration tube, and performing trypsin enzymolysis

(4) Marking the enzymatic peptide fragments obtained from different patient tissues with an iTRAQ reagent according to an optimized flow, mixing, vacuumizing and then carrying out HPLC pre-grading on a sample;

(5) submitting the peptide fragments obtained by HPLC classification to an online two-dimensional liquid chromatography-mass spectrometry (2D-LC-MS/MS) system for complete proteomics quantitative analysis;

(6) screening and confirming specific protein markers of hepatocellular carcinoma patients suffering from macrovascular invasion at high flux and high accuracy through strong later-stage bioinformatics analysis;

(7) the specific protein marker for the invasion of the large blood vessels, which is obtained by proteomic screening, is confirmed in large-queue hepatocellular carcinoma patients by adopting the technical means of western blotting and immunohistochemistry.

In one embodiment, the method for grading vascular invasion in hepatocellular carcinoma (HCC) patients includes the steps of:

(1) obtaining cancer tissues and corresponding paracancerous tissues of a hepatocellular carcinoma patient suffering from vascular invasion by biopsy puncture;

(2) embedding the obtained tissues in paraffin, and then carrying out continuous section, and using the section for immunohistochemical detection of the protein level of the specific protein marker;

(3) according to the detection result, the staining condition of the section is scored;

(4) and combining the clinical information of the patient, grading the vascular invasion condition of the patient, and predicting the large vascular invasion of the patient.

The present invention also provides a method for grading vascular invasion in hepatocellular carcinoma (HCC) patients, comprising the steps of:

(4) and grading the vascular invasion of the patient according to the score by combining the clinical information of the patient and the vascular invasion of the patient.

The present invention also provides a detection kit for the macrovascular invasion of hepatocellular carcinoma (HCC) patients, characterized in that the kit comprises proteins found to be dysregulated in HCC patients with MAV based on proteomic studies.

In one embodiment, the deregulated protein may be one or more of proteins numbered P25325, P05062, P00167, P52758, Q93099, P04424, P11586, Q13228, P08319, P30084, P34913, Q6UX53, Q9UI17, P169930, P00966, P05089, P22033, A6NLP5, P55084, P42765, P31327, O75891, P05091, P09467, Q16822, Q53FZ2, Q9UBR1, Q93088, P00505, P54868, P36871, P0635404, Q3LXA3, P06737, Q9Y2P5, Q50251, P33121, P50225, P08133, P41, O953554, P06573, Q231864, Q0718678, or Q368647 in the Uniprot database.

In one embodiment, the deregulated protein can be one or more of CPS1, ASS1, ASL, ARG1, BHMT, DMGDH, Annexin a6, or CES 1.

In one embodiment, the deregulated protein can be one or more of CPS1, ASL, BHMT, Annexin a6, or CES 1.

The invention also provides application of the detection kit in predicting the great vessel invasion of hepatocellular carcinoma (HCC) patients.

The invention aims to solve the problem of finding and confirming specific protein markers of HCC patients with different degrees of vascular invasion, further elucidating the molecular mechanism of HCC macrovascular invasion and applying the molecular markers to clinical prediction, diagnosis and treatment of HCC macrovascular invasion. The invention develops a detection kit which can be used for immunoblotting and immunohistochemical analysis and provides a method for carrying out molecular typing on HCC with different degrees of vascular invasion. The detection of the level of the protein marker discovered by the invention through the kit can diagnose and grade the vascular invasion condition at the early stage of HCC occurrence, and suggest an optimized treatment scheme for the treatment, thereby reducing the HCC macrovascular invasion occurrence and improving the prognosis and survival rate of HCC.

Brief description of the drawings

FIG. 1 is a workflow of MAV-specific protein marker high-throughput screening of large blood vessel invasion HCC patients.

FIG. 2 identification and quantification of proteins in liver cancer tissue from blood vessels invading HCC patients from itraQ experiments.

FIG. 3. bioinformatics analysis screening for MAV-specific differential proteins in patients with large vessel invasion HCC.

Figure 4 GO and KEGG analysis revealed that urea circulation was significantly down-regulated in cancer tissues of HCC patients with large vessel invasion.

Figure 5. validation of potential MAV-specific protein markers by Western blot.

Figure 6 further validation of potential MAV-specific protein markers by IHC staining.

Figure 7 Western blot and IHC staining indicate that CPS1 is a MAV-specific protein marker, whereas the expression level of CES1 is correlated with the course of vascular invasion.

Figure 8. significant down-regulation of urea cycle is a specific clinically relevant proteomic feature in cancer tissues of HCC patients with large vessel invasion.

Detailed description of the preferred embodiments

The object of the invention can be achieved by the following measures: 1. a total of 61 patients with liver cancer who developed different degrees of vascular invasion (including 23 cases of no vascular invasion, 23 cases of microvascular invasion and 15 cases of macrovascular invasion) and the corresponding paracancerous tissues were obtained by surgery. 2. 2 cases without vascular invasion, 2 cases with microvascular invasion and 4 cases with macrovascular invasion are selected, and different cancer tissues and corresponding para-cancer tissues of patients are subjected to Bioruptor non-contact ultrasonic disruption to efficiently extract total protein. The obtained protein is subjected to reductive alkylation, and is loaded on an ultrafiltration tube for trypsin enzymolysis. 3. And marking the enzymatic peptide fragments obtained from different patient tissues with an iTRAQ reagent according to an optimized flow, mixing, vacuumizing and then carrying out HPLC pre-grading on the sample. 4. And submitting the peptide fragments obtained by HPLC classification to an online two-dimensional liquid chromatography-mass spectrometry (2D-LC-MS/MS) system for complete proteomics quantitative analysis. 5. The specific protein markers in hepatocellular carcinoma patients suffering from macrovascular invasion are screened and confirmed with high throughput and high accuracy through powerful post-bioinformatics analysis, such as PCA analysis, Heatmap, volcano graph, Venn graph, GO analysis, KEGG analysis, TCGA liver cancer database analysis and the like. 6. The specific marker of the great vessel invasion obtained by proteomic screening is verified in 53 large-queue hepatocellular carcinoma patients by using a detection kit containing protein markers and adopting technical means such as protein immunoblotting (Western blotting) and Immunohistochemistry (IHC).

The invention provides a stable and efficient technical route for screening and confirming specific protein markers of large blood vessel invasion from liver cancer patients with different degrees of blood vessel invasion based on disease proteomics (figure 1). In patients with large vessel invasion (MAV), we found that 47 proteins were significantly down-regulated in HCC tumor tissue, as shown in figures 2 and 3. In fig. 2, a. shows the number of proteins identified and quantified in two iTRAQ experiments; principal component analysis of four patient cancers and paracancer samples in itraq experiment 1; principal component analysis of four patient cancers and paracancer samples in itraq experiment 2; hepatomap of four patient cancers and paracancer samples in itraq experiment 1; itraq experiment 2 heatmaps of four patient cancers and paracancer samples. More importantly, 30 of these proteins were unchanged in HCC patients without MAV (figure 3). In FIG. 3, A-D are volcano plot analyses of patients A-D, respectively, with green on the left for down-regulated protein and red on the right for up-regulated protein; E. up-and down-regulating protein profiles in patient a-D cancer tissues; F. wien graph analysis of upregulated proteins in a-D cancer tissues of patients; G. wien graph analysis of downregulated proteins in patient a-D cancer tissues; H. heatmap expressing a protein that undergoes a significant change in the expression of the patient's A-D cancer tissue; wien graph analysis of all stably up-regulated protein (I) and all stably down-regulated protein (J) in eight patients a-H. GO analysis of these 47 significantly down-regulated proteins revealed that the most highly enriched biological process was the urea cycle, indicating that down-regulation of urea cycle is closely linked to macrovascular invasion of HCC (figure 4). In fig. 4, a KEGG analysis of stable down-regulated proteins in cancer tissues of MAV patients; B. analysis of biological processes that stably down-regulate proteins in the cancer tissues of MAV patients; C. the urea cycle is stably down-regulated in the cancer tissues of MAV patients; D. a network of interactions between key enzymes of the urea cycle that are stably down-regulated in the cancerous tissues of MAV patients.

To further verify proteomic results, the present inventors developed a detection kit comprising 9 proteins (CPS1, ASS1, ASL, ARG1, BHMT, DMGDH, Annexin a6, CES1 and CKAP4), which comprises 100 μ l each of the above 9 proteins. The 9 candidate proteins were confirmed by Western blot method to be significantly dysregulated (p <0.01) in HCC patients with large vessel invasion (11 cases), including 8 proteins (CPS1, ASS1, ASL, ARG1, BHMT, DMGDH, Annexin a6 and CES1) down-regulation and 1 protein (CKAP4) up-regulation (fig. 5). Western blot validation of proteins that undergo stable changes in MAV patient cancer tissues in FIGS. 5, A-B; quantification of the Western blot results of the variant proteins in A and B in the cancer tissues of 11 MAV patients. As shown in the data in FIG. 5, the average fold change of the down-regulation of proteins CPS1, ASS1, ASL, ARG1, BHMT, DMGDH, Annexin A6 and CES1 is more than 1.2 times, and the up-regulation of protein CKAP4 is less than 0.8 times. In addition, further validated using immunohistochemical staining, significant down-regulation of CPS1, ASL and ARG1 (key enzymes involved in the urea cycle) and Annexin a6 and CES1 (major proteins regulating cholesterol homeostasis and fatty acid ester metabolism) in large vessel invading patients (11 cases) (figure 6). In fig. 6, a. immunohistochemical validation of stable down-regulated proteins in cancer tissues of MAV patients; B. scoring and statistics of stable down-regulated protein immunohistochemical results in cancer tissues of 11 MAV patients. Meanwhile, the invention confirms that CPS1 is down-regulated only in large vessel invasion HCC patients (p <0.01) in 53 HCC patients (comprising 21 patients without vessel invasion, 21 patients with microvascular invasion and 11 patients with large vessel invasion) and CES1 is gradually reduced with the increase of the severity degree of the blood vessel invasion of the HCC patients (p <0.01), so that grading of the HCC blood vessel invasion can be realized according to the protein expression levels of CPS1 and CES1 (figure 7). Immunohistochemical (a) and Western blot (B) of cps1 and CES1 protein expression levels in different blood vessel invasion HCC patient tissues in fig. 7; C. statistical results for B-picture. Finally, it was observed in proteomic results that all 5 key enzymes (CPS1, OTC, ASS1, ARG1 and ASL) of the whole urea cycle and 5 metabolic enzymes upstream thereof (BHMT, BHMT2, DMGDH, SARDH and GLDC) were significantly down-regulated in tumor tissues of HCC patients with MAV (p < 0.05); further using TCGA liver cancer database large-queue analysis, it was found that down-regulation of mRNA levels of these urea cycle-associated enzymes (CPS1, OTC, ALDH7a1, BHMT2, DMGDH and SARDH) significantly reduced the overall survival (p <0.05) of HCC patients (182) (fig. 8). In fig. 8, a.itraq proteomics results revealed that key enzymes of the urea cycle and their upstream pathway proteins were stably down-regulated in the cancer tissues of MAV patients; tcga hepatoma database large-queue analysis found that down-regulation of mRNA levels of urea cycle-associated enzymes (CPS1, OTC, ALDH7a1, BHMT2, DMGDH and SARDH) significantly reduced the overall survival of HCC patients (182) (p < 0.05). Thus, the present invention reveals a molecular mechanism by which urea cycle disturbances may reduce survival in HCC patients by causing macrovascular invasion and tumor migration; the significant down-regulation of urea cycle is a specific clinically relevant proteomic feature in HCC patients with large vessel invasion, can be used for accurate diagnosis of MAV, and provides a potential therapeutic target for treatment of MAV.

1. Method of producing a composite material

Acquisition of tissue samples from HCC patients

The study was approved by the ethical committee of the affiliated drumbeat hospital of the medical college of Nanjing university. All the tissues of HCC patients were from hepatobiliary surgery, a drumbeat hospital affiliated with the university of medicine, Nanjing. All the general and clinical information of the participating patients is recorded and archived in detail. All patients were informed and consented to their tissue samples for the study. After liver tissue samples were obtained from HCC patients, liver tissue was snap frozen in liquid nitrogen and stored at-80 ℃ for further analysis. For iTRAQ experiments, HCC patients with MAV (4 patients a-D), MVI (+) (2 patients E, F) and MVI (-) (2 patients G, H) were selected based on clinical classification of HCC patients, while pathologically confirmed liver cancer tissues and adjacent paracancerous liver tissues from the same patient were collected.

Extraction, enzymatic digestion and iTRAQ labeling of liver proteins

100mg of liver cancer tissue or adjacent para-cancerous tissue from HCC patients was used for protein extraction. Briefly, tissues were first homogenized in RIPA lysis buffer (50mM Tris-HCl, 0.5% NP-40, 0.25% sodium deoxycholate, 1mM EDTA and 1% protease inhibitor cocktail III and V, 1mM PMSF, 1mM NaVO3, 1mM NaF, pH 7.4). Incubate on ice for 30 minutes, then perform non-contact sonication lysis at 4 ℃ using a Bioruptor Plus sonication device (Diagenode, Belgium). The supernatant was collected and centrifuged at 12000rpm for 30 minutes to remove cell debris. Protein concentration was determined with BCA protein assay kit and further confirmed by coomassie brilliant blue staining.

The extracted proteins were subjected to ultrafiltration tube enzymatic digestion, 100. mu.g of total protein was diluted to 100. mu.l with 0.5M TEAB, reduced with 5mM CEP for 1 hour at 55 ℃ and then alkylated with 6.25mM MMTS for 30 minutes at room temperature in the dark. The obtained protein was then transferred to a 10K ultrafiltration tube (Vivacon, USA), centrifuged at 12000rpm for 30 minutes to remove the solvent, and washed 3 times with 100. mu.l of 0.5M TEAB by repeated centrifugation. Finally, trypsin (Promega, USA) was added to the ultrafiltration tube membrane in a 1:50 trypsin to protein mass ratio, the first enzymolysis was overnight, and a 1: 100 trypsin was used for a second 4 hours of enzymatic digestion at 37 ℃.

Table 1: iTRAQ laboratory patient and different sample marker information

The resulting peptide fragments were collected and labeled with the iTRAQ Reagent-8plex Multiplex kit (abciex, u.k.limited) according to the manufacturer's instructions. The sample labeling is shown in table 1: in iTRAQ experiment 1, 4 HCC paracancerous samples (P) from patients a-D were labeled with iTRAQ tags 113,115,117, or 119; 4 HCC tumor samples (T) from patients A-D were labeled with iTRAQ tags 114,116,118 or 121. In

iTRAQ experiment

2, 4 HCC paracancerous samples (P) from patient E-H were labeled with iTRAQ tags 113,115,117, or 119. 4 HCC tumor samples (T) from patients E-H were labeled with iTRAQ tags 114,116,118 or 121. For each iTRAQ experiment, all 8 labeled peptides were mixed together, flash dried, and purified using a high performance liquid chromatography (HPLC, SHIMADZU) system and a Durashell C18 column (5 μm,

4.6 × 250mm) into 48 samples finally, the 48 samples were combined into 16 samples and after desalting and Speedvac drying, the 16 samples were resuspended in 3% (v/v) formic acid 2% (v/v) acetonitrile for LC-MS/MS analysis.

LC-MS/MS analysis

MS data collection was performed with NanoLC.2D (Eksient Technologies) and TripleTOF 5600 system (AB SCIEX, Concord, ON.) samples were chromatographed using a 90 minute gradient from 2-80% (mobile phase A: 0.1% (v/v) formic acid, 2% (v/v) acetonitrile; mobile phase B: 0.1% (v/v) formic acid, 98% (v/v) acetonitrile). after sample application to NanoLC column 3C18-CL, 75 μm × cm (Eksient Technologies), the elution gradient of the peptide fragments included an increase in solvent B concentration from 2% to 22% over 60 minutes, followed by an increase from 22% B to 35% B over 18 minutes, then a ramp to 80% B over 6 minutes, maintained at 80% B over the last 6 minutes, all operations maintained a constant flow rate of 300 nL/min. in the 350,500 m/z range of the ion charge-trapping range, MS was used to select the precursor for ion trapping in the ion charge trapping range of 250/5 MS 632, and the ion trapping range of 100 sec for trapping of the precursor was performed at 100 sec, 100 sec for trapping range of 100 sec of the ion charge trapping range of the MS 632.

Database search

Raw MS data were submitted to ProteinPilot software (version 4.5, AB Sciex) for data analysis. MS/MS data were searched against Human Sapiens in the UniProt database (2016, 9/4/2016, containing 160,566 sequences, http:// www.uniprot.org/proteins/UP 000005640). The following search parameters were used: the apparatus is TripleTOF 5600, iTRAQ quantification, using MMTS modified cysteine, biological modification was selected as the focus of protein identification, quantification, trypsin enzymatic hydrolysis, bias correction and background correction to check protein quantification and normalization. For False Discovery Rate (FDR) calculations, an automated decoy database search strategy is used to estimate FDR using a PSPEP (proteomics system performance evaluation pipeline software) algorithm. Only proteins with valid p-values were selected for further analysis. To identify proteins that are significantly differentially expressed in cancer/paracancer, significant upregulation was considered when the fold change in protein was >1.2 fold and the p-value was < 0.05; significant downregulation was considered when fold change <0.8 fold and p-value < 0.05.

Bioinformatics analysis

The similarity or heterogeneity of all tumors and paraneoplastic tissues from 4 patients in the same iTRAQ experiment was determined by PCA analysis and thermography of all quantified proteins. The overall dynamic change in protein expression levels (T/P) between tumor and paraneoplastic tissues of each patient was analyzed using volcano plots drawn from "ggplot 2" in the R language package. The reproducibility of the potential differentially expressed proteins in each patient group was analyzed by the R language package "VennDiagram". Proteins stably differentially expressed in tumor and paraneoplastic tissues were demonstrated by heat map (package R language "phetmap") and classified by Gene Ontology (GO) and Kyoto Encyclopedia of genes and genomics (KEGG pathway) annotation using the DAVID online tool (http:// DAVID. For each class, the two-tailed Fisher exact test was used to test the enrichment of differentially expressed proteins for all identified proteins. GO and KEGG pathways with corrected p values <0.05 were considered significantly enriched. The KEGG database was used to map differentially expressed proteins (https:// www.kegg.jp) on relevant metabolic pathways. Protein-protein interaction network analysis was performed by STRING on-line tool (http:// STRING-db. org) and manually recombined.

Western blot analysis

The present invention develops a kit comprising proteins found to be dysregulated in HCC patients with MAV based on proteomic studies. The deregulated protein may be one or more of proteins numbered P25325, P05062, P00167, P52758, Q93099, P04424, P11586, Q13228, P08319, P30084, P34913, Q6UX53, Q9UI17, P169930, P00966, P89, P22033, A6NLP5, P55084, P42765, P31327, O75891, P05091, P09467, Q16822, Q53FZ 050 2, Q9UBR1, Q93088, P50205, P54868, P36871, P35573, P80404, Q3LXA3, P06737, Q9Y2P5, Q16851, P33121, P50225, P23186833, P23141, O954, P9554, P957, Q4594, W0628678, Q06278, or Q368647 in the Uniprot database. The deregulated protein may also be one or more of CPS1, ASS1, ASL, ARG1, BHMT, DMGDH, Annexin A6, CES1 and CKAP 4. The present invention detects and verifies the expression of 9 proteins in patients with great vessel invasion HCC by Western blot analysis with a detection kit comprising 9 proteins (100. mu.l each of CPS1, ASS1, ASL, ARG1, BHMT, DMGDH, Annexin A6, CES1 and CKAP4 antibodies). 11 patients with large vessel invasion of HCC were selected based on clinical information and their cancerous tissues and adjacent paracancerous tissues were homogenized in a protease inhibitor cocktail (Roche Applied Science) containing 0.5% SDS. 100 μ g of protein from each sample was separated by 12% SDS-PAGE gel and transferred to PVDF membrane (Merck Millipore, Darmstadt, Germany). Membranes were blocked with 5% skim milk in TBST and incubated with primary antibody overnight at 4 ℃. The primary antibody used was as follows: carbamoyl phosphate synthetase 1(CPS1, 1: 2500,18703-1-AP, Proteintetech), argininosuccinate synthetase 1(ASS1, 1: 5000,16210-1-AP, Proteintetech), argininosuccinate lyase (ASL, 1: 800,16645-1-AP, Proteintetech), arginase 1(ARG1, 1: 2500,16001-1-AP, Proteintetech), betaine-homocysteine methyltransferase (BHMT, 1: 2000,15965-1-AP, Proteintetech), dimethylglycine dehydrogenase (DMGDH, 1: 5000,24813-1-AP, Proteitech), carboxyesterase (CES1, 1: 2500,14587-1 AP, Proteitech), annexin A6 (AnnexinnA 6, 1: 1000,12542-1-AP, Teintech) and cytoskeleton-related protein 4 (ProteinAP 4, AP 1: 8000,16686-8000,16686). After washing with TBST, the membrane was incubated with horseradish peroxidase (HRP) conjugated secondary antibody for 1 hour at room temperature and exposed using a chemiluminescent HRP substrate (Merck Millipore, Darmstadt, Germany). The results show that CPS1, ASS1, ASL, ARG1, BHMT, DMGDH, Annexin a6 and CES1 were significantly reduced in large vessel invasion of HCC cancer tissue (p <0.01), while CKAP4 was significantly increased (p < 0.01).

Immunohistochemical staining

In order to use the protein markers related to the macrovascular invasion verified in the above experiments for grading the vascular invasion condition of hepatocellular carcinoma patients and prediction of macrovascular invasion, we applied the kit of the present invention to perform immunohistochemical staining. Liver cancer and tissues adjacent to the cancer were examined from 53 HCC patients (including 11 MAV patients, 21 MVI (+) patients and 21 MVI (-) patients). The kit is used as follows: formalin-fixed, paraffin-embedded liver cancer and para-carcinoma tissues from HCC patients were serially sectioned (5 μm thick) and IHC stained using antibodies specific for CPS1(1:50), ASL (1: 250), BHMT (1: 100), Annexin a6 (1: 250) and CES1(1: 40). After washing with wash buffer, the slides were incubated with an appropriate HRP-conjugated secondary antibody. After staining was complete, the expression levels of protein on the slide were scored as negative (0), weak (1+), moderate (2+) and strong (3+) according to staining intensity. The results indicate that expression levels of CPS1, ASL, BHMT1, Annexin a6 and CES1 are significantly reduced (0 or 1+) in large vessel invading HCC cancer tissues; meanwhile, CPS1 is only down-regulated (0 or 1+) in a patient with large blood vessel invasion, while CES1 gradually decreases with the increase of the severity of the blood vessel invasion of the patient with HCC (the large blood vessel invasion is 0 or 1+, the micro blood vessel invasion is 2+, and the blood vessel invasion is 3+) so that the grading of the HCC blood vessel invasion can be realized according to the protein expression levels of CPS1 and CES 1. The specific classification criteria are as follows: 1. when the staining intensity scores of CPS1, ASL, BHMT1, Annexin A6 and CES1 are all 0 or 1+, the macrovascular invasion is recommended to be graded; 2. when the staining intensity scores of CPS1, ASL, BHMT1 and Annexin a6 are both 2+ or 3+ while the staining intensity score of CES1 is 2+, the rating is recommended as microvascular invasion; 3. when the staining intensity scores of CPS1, ASL, BHMT1, Annexin A6 and CES1 are all 3+, it is recommended to grade as no vascular invasion.

Clinically, the conventional means for detecting and diagnosing the great vessel invasion of HCC patients are imaging means, and they have the disadvantage that a definite image can be formed only when the great vessel invasion of liver cancer has occurred, so that diagnosis can be made. The kit of the present invention has the advantage that it can be used to stratify the vascular invasion of HCC patients at a molecular level and to directly predict whether a patient is likely to develop macrovascular invasion. Even if HCC patients are in a stage without vascular invasion or microvascular invasion, the kit can predict the possibility of liver cancer macrovascular invasion by detecting the specific protein marker, so as to intervene as early as possible and prevent the macrovascular invasion, and has very important significance for improving the survival rate and prognosis of liver cancer patients.

Statistical analysis

All data were processed using SPSS12.0(SPSS inc., Chicago, IL), experimental data are shown as mean ± standard deviation (s.d.), and p <0.05 is considered statistically significant. Paired T-tests were used to compare whether differences in protein levels between paired cancer and para-cancer tissues were significant.

Claims

1. A method for predicting macrovascular invasion in a hepatocellular carcinoma (HCC) patient, characterized by predicting macrovascular invasion in a hepatocellular carcinoma (HCC) patient when one or more specific protein markers, which are proteins found to be dysregulated based on proteomic studies in HCC patients with MAV, are dysregulated, i.e. expression level up-or down-regulated.

2. The method of claim 1, wherein the dysregulation of specific protein markers is the down-regulation of the expression of one or more of P25325, P05062, P00167, P52758, Q93099, P04424, P11586, Q13228, P08319, P30084, P34913, Q6UX53, Q9UI17, P169930, P00966, P05089, P22033, A6NLP5, P55084, P42765, P31327, O75891, P05091, P09467, Q16822, Q53FZ2, Q9UBR1, Q93088, P00505, P54868, P36871, P3535404, Q3LXA3, P06483, Q9Y2P 63, Q16851, P1686751, P1689551, P50247, P07147, P08147, P0814, P08147, P0814, P0813 x 7375, P05025, P0813.

3. The method for predicting large vessel invasion of hepatocellular carcinoma (HCC) patient as claimed in claim 1 wherein said deregulated expression level of specific protein markers is the down-regulation of expression level of one or more of CPS1, ASS1, ASL, ARG1, BHMT, DMGDH, Annexin a6 and CES 1.

4. The method for predicting large vessel invasion in hepatocellular carcinoma (HCC) patient according to claim 1 wherein said specific protein marker disregulation is up-regulation of CKAP4 expression level.

5. The method for predicting large vessel invasion in hepatocellular carcinoma (HCC) patient as claimed in claim 1 wherein said specific protein marker deregulation is the down-regulation of the expression level of one or more of the 5 key enzymes of urea cycle CPS1, OTC, ASS1, ARG1 and ASL.

6. The method for predicting large vessel invasion in hepatocellular carcinoma (HCC) patient according to claim 1 wherein said specific protein marker dysregulation is the down-regulation of the expression level of one or more of the 5 metabolic enzymes BHMT, BHMT2, DMGDH, SARDH and GLDC upstream of the urea cycle.

7. The method for predicting the macrovascular invasion of a hepatocellular carcinoma (HCC) patient according to claim 1 characterized in that said specific protein markers are determined by the following steps:

8. The method for predicting large vessel invasion in hepatocellular carcinoma (HCC) patient according to claim 1, characterized in that it comprises the following steps:

9. A method for grading vascular invasion in hepatocellular carcinoma (HCC) patients comprising the steps of:

10. A detection kit for the macrovascular invasion of hepatocellular carcinoma (HCC) patients, characterized in that said kit comprises proteins found to be dysregulated in HCC patients with MAV based on proteomic studies.

11. The detection kit for the great vessel invasion of hepatocellular carcinoma (HCC) patient according to claim 11, characterized in that said protein markers comprise one or more of the 5 key enzymes of the urea cycle CPS1, OTC, ASS1, ARG1 and ASL.

12. Use of a test kit according to any one of claims 12-14 for predicting large vessel invasion in a patient with hepatocellular carcinoma (HCC).